Figure 6.

MK induces NET formation via LRP1. (a) Representative immunofluorescence images of BM-derived isolated mPMN. Cells were exposed to sMK, iMK, PMA or left untreated for control (w/o) on murine fibrinogen for 16 h. DNA (white) and H3Cit (magenta). Arrows indicate NETs. Bar, 40 µm. n = 3. (b) Images of human PMNs freshly isolated from healthy donors. Cells were stimulated in the presence of human fibrinogen with sMK, iMK, or PMA or left unstimulated for control (w/o). Staining for DNA (white) and MPO (green) was subsequently conducted. n = 3. (c) Representative immunofluorescence images of dHoxb8-LRP1^ctrl or dHoxb8-LRP1^cKO cells. Cells were stimulated in the presence of iMK, PMA, or left unstimulated for control (w/o). Staining for DNA (white) and H3Cit (magenta) is depicted as indicated. Bar, 40 µm. n = 3. (d) Quantitative analysis of NET formation of dHoxb8-LRP1^ctrl or dHoxb8-LRP1^cKO in the presence of iMK (dHoxb8-LRP1^ctrl, 21.3 ± 1.9 NET⁺ cells [%]; dHoxb8-LRP1^cKO, 5.12 ± 1.7 NET⁺ cells [%]), PMA (dHoxb8-LRP1^ctrl, 51.1 ± 2.7 NET⁺ cells [%]; dHoxb8-LRP1^cKO, 50.9 ± 3.6 NET⁺ cells [%]), or without simulation (dHoxb8-LRP1^ctrl, 6.2 ± 1.4 NET⁺ cells [%]; dHoxb8-LRP1^cKO, 3.2 ± 0.6 NET⁺ cells [%]). Data show the number of NET⁺ cells as a percentage of all dHoxb8 cells. n = 3. To determine P values, one-way ANOVA with Holm–Sidak multiple comparisons test was performed. ***, P < 0.001. Data are presented as mean ± SEM.